Porcine β-Defensin 2 Attenuates Inflammation and Mucosal Lesions in Dextran Sodium Sulfate−Induced Colitis

This information is current as Feifei Han, Haiwen Zhang, Xi Xia, Haitao Xiong, Deguang of September 25, 2021. Song, Xin Zong and Yizhen Wang J Immunol 2015; 194:1882-1893; Prepublished online 19 January 2015; doi: 10.4049/jimmunol.1402300

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Porcine b-Defensin 2 Attenuates Inflammation and Mucosal Lesions in Dextran Sodium Sulfate–Induced Colitis

Feifei Han,*,†,1 Haiwen Zhang,*,1 Xi Xia,* Haitao Xiong,* Deguang Song,* Xin Zong,* and Yizhen Wang*

Intestinal permeability plays a critical role in the etiopathogenesis of ulcerative colitis. Defensins, including porcine b-defensin (pBD)2, are crucial antimicrobial for gut protection owing to their antibacterial and immunomodulatory activities. The purpose of this study was to investigate the protective effects of pBD2 on mucosal injury and the disruption of the epithelial barrier during the pathological process of dextran sodium sulfate (DSS)–induced colitis. The effects and mechanism of pBD2 were evaluated both using a DSS-induced C57BL/6 mouse model and, in vitro, using Caco-2 and RAW264.7 cells. DSS-induced colitis was characterized by higher disease activity index, shortened colon length, elevated activities of and peroxidase, histologic evidence of inflammation, and increased expression levels of TNF-a, IL-6, and IL-8. pBD2 increased the Downloaded from expression of zonula occludens-1, zonula occludens-2, claudin-1, mucin-1, and mucin-2 mRNA and , and it decreased permeability to FITC-D, as well as apoptosis, in DSS-treated mice. pBD2 also decreased inflammatory infiltrates of the colon . In Caco-2 cells, pBD2 increased transepithelial electrical resistance and mucin mRNA expression, and it decreased the permeability of FITC-D while preserving the structural integrity of the tight junctions. The effects of pBD2 appeared to be through upregulation of the expression of genes associated with tight junctions and mucins, and by suppressing DSS-induced increases in inflammation, inducible NO synthase, cyclooxygenase-2, and apoptosis. These results show that pBD2 improves DSS- http://www.jimmunol.org/ induced changes in mucosal lesions and paracellular permeability, possibly by affecting the activation of NF-kB signaling. The present study demonstrates that intrarectal administration of pBD2 may be a novel preventive option for ulcerative colitis. The Journal of Immunology, 2015, 194: 1882–1893.

lcerative colitis (UC), a subcategory of inflammatory flammation and relapses of disease activity are associated with bowel disease (IBD), is a chronic inflammatory disease engagement of the innate and adaptive immune responses, in- U characterized by aberrant immune responses to luminal cluding increased production of TNF-a and IFN-g in the intestine in genetically susceptible subjects (1). Relapsing/remitt- (4, 5). These proinflammatory cytokines are potential pathogenic by guest on September 25, 2021 ing episodes of UC are associated with marked variations in the factors that impair mucosal barrier function and intestinal per- production of proinflammatory cytokines (2, 3). Various mecha- meability. A disproportionately large number of first-degree rela- nisms have been proposed for UC, including an inappropriate tives of patients with IBD have increased intestinal permeability. inflammatory response to , autoimmunity, and an ab- This suggests that barrier dysfunction may be an early defect. normal immune response to microbiota or dietary Ags. Although Previous studies have demonstrated a decreased expression of the exact pathogenesis of IBD is unknown, the initiation of in- junction complex proteins in the intestinal mucosa of patients with IBD (6, 7). Impaired gut epithelial barrier function may lead to *Key Laboratory of Animal Nutrition and Feed Science in Eastern China, Ministry of persistent immune reactions, thus augmenting gut inflammation Agriculture, College of Animal Sciences, Zhejiang University, Hangzhou 310058, (8). The intestinal epithelial barrier consists of epithelial cells † China; and Food Safety Key Laboratory of Zhejiang Province, School of Food and intercellular junctions (9). Tight junctions (TJs) are the most Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310018, China apical intercellular structure in epithelial cells, accounting for 1F.H. and H.Z. contributed equally to this work. cell–cell adhesion, polarity, and a permeability barrier to the para- cellular transport of solutes (10). TJs create a semipermeable Received for publication September 8, 2014. Accepted for publication December 9, 2014. barrier separating different organ compartments. TJs are com- This work was supported by National Natural Science Foundation for Distinguished posed of a large number of components, such as occludins, Young Scholars of China Grant 31025027, National Natural Science Foundation of claudins, and zonula occludens (zo)-1. Both claudins and occlu- China Grant 31201809, and Earmarked Fund for Modern Agro-industry Technology dins are coupled either directly or indirectly to cytoskeleton actin Research System of China Grant CARS-36. filaments with zo-1 and other factors. The distribution of claudins Address correspondence and reprint requests to Prof. Yizhen Wang, College of Animal Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, varies along the gut epithelia, and they are differentially expressed China. E-mail address: [email protected] in connecting cells through the formation of zipper structures (11, The online version of this article contains supplemental material. 12). Once the mucosal barrier is breached, the submucosa is ex- Abbreviations used in this article: BW, body weight; CD, Crohn’s disease; COX-2, posed to a vast pool of luminal Ags, including food and bacteria, cyclooxygenase-2; DAI, disease activity index; DSS, dextran sodium sulfate; EPO, and the innate immune responses are engaged to produce large ; hBD, human b-defensin; IBD, inflammatory bowel disease; iNOS, inducible NO synthase; mBD, mouse b-defensin; MPO, myeloperoxidase; amounts of cytokines. Therefore, maintaining TJs and barrier func- OB, occult blood; pBD, porcine b-defensin; PD, potential difference; TEER, trans- tion may provide benefits in the adjuvant therapy of many gastro- epithelial electrical resistance; TJ, tight junction; UC, ulcerative colitis; zo, zonula intestinal diseases, including IBD (13, 14). occludens. In the gastrointestinal tract, the first line of defense against Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 invading pathogens is provided by the innate (15). www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402300 The Journal of Immunology 1883

Part of this immune response is the release of The purpose of this experimental design is to investigate the prophylactic into the lumen of the intestinal tract. These peptides are capable of effect of pBD2 on inflammation and mucosal lesions in DSS-induced colitis. directly killing a wide variety of bacterial and viral pathogens. As As for the mode of administration, we have tested the distribution in the mouse body of pBD2 after intrarectal administration of FITC-labeled pBD2 a family of antimicrobial peptides, defensins are secreted by various in the preliminary experiment. We found that the FITC-labeled pBD2 was cells as a component of the innate host defense (16). These peptides distributed throughout the body 10 min after injection and disappeared have bactericidal activity by forming micropores in the phospho- practically 24 h after injection (Supplemental Fig. 1). Therefore, rectal lipid bilayer of bacterial membranes, causing the loss of structural administration was selected in this experiment. The mice were divided into four groups. The control and pBD2-treated groups were administered PBS integrity and collapse of the bacterial cell (17). This antimicrobial or 5 mg/kg pBD2 rectally without DSS. The DSS and DSS plus pBD2 activity allows defensins to protect the host epithelium and stem groups were administered PBS or 5 mg/kg pBD2 rectally, together with cells from virulent pathogens, and it also helps to regulate the oral administration of 3.5% DSS. pBD2 or PBS were administered intra- number and composition of commensal microbiota (18). rectally once daily for 7 consecutive days. Mice were kept in an inverted Increasing evidence suggests that decreased levels or malfunc- position for 1 min after each administration to prevent leakage from the anus. Mice were euthanized at day 7 by cervical dislocation and blood and tioning of b-defensins in the intestine might lead to a disturbance tissues were collected. Colons were separated from the proximal rectum, of homeostasis, resulting in intestinal diseases such as Crohn’s close to their passage under the pelvisternum. The colon length, as an index disease (CD) and UC (19). The expression of human b-defensins of disease, was measured between the ileocecal junction and the proximal (hBD-1 and hBD-2) is altered in patients suffering from UC but not rectum, and the weight was determined (25). Some colonic tissues were excised and homogenized in RIPA lysis buffer. Equal amounts of protein CD (20). Immunohistochemistry showed that hBD-2 is barely (40 mg/lane) were subjected to Western blotting and ELISA. Some colonic present in the uninflamed colon, but it is induced during inflam- tissues were also used for H&E staining and TUNEL assays as described mation. The lower expression of hBD-2 in CD compared with UC previously (26). Other tissues were stored at 270˚C for further analyses. Downloaded from indicates different responses of the mucosal innate defense to these Assessment of disease activity two disorders. Furthermore, an upregulation of intestinal defensins is observed as an early response to inflammation. The Rachmilewitz disease activity index (DAI) was assessed by an inves- tigator blinded to the protocol according to a standard scoring system (27). b-Defensins exert their anti-inflammatory effects by inhibiting Body weight (BW), stool consistency, and occult blood (OB) in the stool the production of proinflammatory cytokines (such as TNF-a) and were recorded daily. Loss in BW was scored as: 0, no weight loss; 1, weight . interfering with MAPK and NF-kB signaling pathways (21–23). A loss of 1–5% from baseline; 2, 5–10%; 3, 10–20%; and 4, 20%. For stool http://www.jimmunol.org/ previous study tested the influence of b-defensins on ulcer healing consistency, a score of 0 was assigned for well-formed pellets, 2 for pasty in humans. It was discovered that b-defensins were overexpressed and semiformed stools that did not adhere to the anus, and 4 for liquid stools that adhered to the anus. For OB, a score of 0 was assigned for no blood, 2 in diabetic foot ulcers. This suggested that b-defensins could for positive OB, and 4 for gross bleeding. These scores are added together promote (24). These findings seem to indicate that and divided by three, resulting in DAIs ranging from 0 (healthy) to 4 b-defensins play a crucial role in the development and healing of (maximal colitis activity). BW loss was calculated as the percentage dif- mucosal damage and inflammation in UC. ference between the original BW (day 0) and the BW on any particular day. The association of b-defensins with dextran sodium sulfate Histology and immunohistochemistry (DSS)–induced acute colitis is unknown. Thus, the present study The distal colon was taken for histological analysis. Samples were fixed in by guest on September 25, 2021 sought to elucidate the efficacy of porcine b-defensin (pBD)2 in 10% buffered formalin overnight and kept in 70% ethanol until processed. prevention of colitis. pBD2 was administered intrarectally to mice Tissues were embedded in paraffin and cut into 5-mm-thick sections. For and a parallel treatment starting together with DSS. We hypoth- each sample, sections were stained with H&E and mounted in Permount esized that pBD2 could exert its anti-inflammatory effect on colitis (Fisher Scientific, Philadelphia, PA). The mucus-containing cells were stained purple-red. Morphometric analyses of colons were performed using by inhibiting activation of the NF-kB signaling pathway. The an image analysis program (28). preventive effects, as well as the mechanism of action of pBD2 on colitis, were investigated by assessing alterations in the integrity Assessment of apoptosis of the intestinal barrier and the innate immune response during The TUNEL method was used to stain apoptotic cells (29). The number of colitis. The results will provide experimental evidence that pBD2 apoptotic cells was counted in four to six randomly selected fields at 3400 is a possible preventive treatment for the pathogenesis of UC. magnification. Measurement of Ig concentration Materials and Methods Concentrations of IgA, IgG, and IgM in sera were determined by ELISA synthesis quantitative kits (eBioscience, San Diego, CA) according to the manu- Linear pBD2 (a 37-aa peptide: DHYICAKKGG TCNFSPCPLF facturer’s instructions. NRIEGTCYSG KAKCCIR) was synthesized by standard solid-phase Enzyme activity measurements procedures with 9-fluorenylmethoxycarbonyl using an Apex 396 peptide synthesizer (Aapptec, Louisville, KY). Ninety-five percent purity of syn- The activities of myeloperoxidase (MPO) and eosinophil peroxidase (EPO) thetic peptides was achieved and verified using Agilent 1200 series HPLC were assessed in colon samples as described previously (30). Results are (Agilent Technologies, Santa Clara, CA) and a Thermo Finnigan LCQ ion expressed in arbitrary units (based on absorbance) per 100 mg tissue. trap mass spectrometer (Thermo Finnigan, San Jose, CA). Peptides were dissolved in PBS for rectal administration and in PBS prepared with Measurement of transepithelial electrical resistance endotoxin-free water for addition to cultured cells. Electrophysiological parameters were measured using a multichannel Animal model voltage/current clamp (model VCC MC6; Physiologic Instruments). Ussing chambers were equipped with two pairs of Ag/AgCl electrodes connected Male 6- to 8-wk-old C57BL/6 mice were obtained from the Laboratory to the chambers via 3 M KCl/3.5% agar bridges for measuring the poten- Animal Center of Zhejiang University (Hangzhou, China) and maintained in tial difference (PD) and for passing current (I). The experiments were per- plastic cages under standard conditions. Standard pelleted diets (Laboratory formed under open circuit conditions where the current was set to 0 and the Animal Center of Zhejiang University, Hangzhou, China) were supplied ad natural transepithelial PD could be observed. For each measurement, the PD libitum. Animal studies were approved by the Institutional Animal Care and value was clamped to 20 mV and the necessary current was recorded. Use Committee of Zhejiang University and performed in accordance with Electrical resistance was determined according to Ohm’s law, that is, R = institutional guidelines. Acute colitis was induced through the oral intake of (20 mV 2 PD)/I. The fluid resistance measured prior to mounting the 3.5% DSS (w/v, molecular mass of 36,000–50,000 Da; MP Biomedicals, tissue in the diffusion chamber system was subtracted, and the net elec- Solon, OH) in fresh tap water ad libitum for 7 d (n = 12/group). No major trical resistance of the colonic epithelium was multiplied by the apparent differences in water consumption were detected between groups. exposed tissue area (0.07 cm2) to yield transepithelial electrical resistance 1884 b-DEFENSIN 2 ATTENUATES INJURY OF COLITIS

(TEER). The effective exposed tissue area (the total area of folded mucosa) 60 min, and terminated at 70˚C for 10 min. Real-time PCR reactions were was unknown, but it was much larger than the apparent exposed tissue conducted in triplicate on a StepOne real-time PCR system (ABI Step- area. To ensure comparability of TEER measurements and sufficient data OnePlus; Applied Biosystems, Foster City, CA) using the GoTaq qPCR repeatability, the differences between the effective exposed area of the Master Mix kit (Promega). The PCR primer sequences were designed epithelium and the apparent exposed tissue area were normalized by pre- using Primer Premier 5.0 and are listed in Table I. Real-time PCR reactions senting all measurements as a percentage of the TEER value at the end of were performed as follows: a precycling stage at 95˚C for 30 s, then 40 the equilibration period for each tissue insert. The ratio of normalized cycles of denaturization at 95˚C for 10 s and annealing at 60˚C for 34 s. TEER of epithelia in different groups was calculated for each time point Fluorescence was measured at the end of each annealing step, and the together with its 90% two-sided confidence interval (corresponding to 95% melting curves were monitored to confirm the specificity of the PCR one-sided confidence interval). products. The 22DDCT method was used to determine the mRNA expres- sion levels (32). Cell culture Immunofluorescence analysis of TJ proteins RAW264.7 and Caco-2 cells were cultured in RPMI 1640 or DMEM medium, respectively, supplemented with 10% heat-inactivated FBS and Colon sections were incubated with a mouse monoclonal anti–claudin-1 glutamine at 37˚C under 5% CO2. For each experiment, cells were de- Ab and an anti–zo-1 Ab (Santa Cruz Biotechnology, Dallas, TX), then 6 tached with a cell scraper. At the cell density used (1 3 10 cells/ml), the with a tetramethylrhodamine isothiocyanate–conjugated secondary Ab. proportion of dead cells was ,1% according to t trypan blue dye exclusion The sections were then washed, mounted in medium containing 40 mg/ml tests. DAPI, and examined with a Leica fluorescence microscope (Keyence, Osaka, Japan). Measurement of NO and PGE2 6 Western blot analysis After incubation of RAW264.7 cells (1 3 10 cells/ml) for 24 h, cells were treated with LPS (1 mg/ml) for 12 h and then further incubated with pBD2 The concentrations of proteins in extracts from RAW264.7 cells were Downloaded from (12.5 mg/ml) for 4 h. The effect of this peptide on NO production was determined using the Coomassie brilliant blue assay. Extracts containing determined by analyzing NO levels with the Griess assay. The effect of equal quantities of proteins (100 mg) were electrophoresed in polyacryl- pBD2 on PGE2 was determined by treating cells with LPS (1 mg/ml) and amide gel. Subsequently, the separated proteins were transferred onto pBD2 (12.5 mg/ml) for 24 h and analyzing PGE2 levels with ELISA as a polyvinylidene difluoride membrane. The membrane was blocked for described previously (31). nonspecific binding for 30 min (5% skimmed milk in PBS) and then in- Measurement of cytokine concentrations cubated overnight at 4˚C with Abs for c-Jun, p–c-Jun, NF-kB p65, NF-kB

p–p65, Akt, p-Akt, IkB-a, or p–IkB-a (Santa Cruz Biotechnology or http://www.jimmunol.org/ Tissue was homogenized and centrifuged for 15 min at 600 3 g at 4˚C. Epitomics, Burlingame, CA). Blots were developed with ECL detection Supernatants were collected for the cytokine assay. The levels of cytokines reagents (Santa Cruz Biotechnology), exposed on Kodak Xdmat blue XB-1 (TNF-a, IL-6, and IL-8) in supernatants were quantified using ELISA kits film, and quantified by BandScan 5.0 software using b-actin as the internal according to the protocol supplied by the manufacturer (MultiSciences control. Biotech, Hangzhou, China). Plates were coated with mAbs overnight and washed. The supernatants were added to the plates and incubated over- Statistical analysis night. Biotinylated Abs were then added and incubated for 1 h. The color All data are expressed as means 6 SEM. Statistical significance was reaction was developed with o-phenylenediamine and H2O2 substrate in assessed by one-way ANOVA followed by Duncan’s test using SPSS 18.0. sodium citrate buffer. The reaction was terminated by the addition of A p value , 0.05 was considered statistically significant. H2SO4. The absorbance was measured at 490 nm using a multimode mi- croplate reader (SpectraMax M5; Molecular Devices, Sunnyvale, CA). by guest on September 25, 2021 Results mRNA analysis by real-time PCR pBD2 ameliorated the clinical symptoms and colonic Total RNA was isolated from intestinal samples or Caco-2 cells by using inflammation during the pathological process of DSS-induced TRIzol (Invitrogen) according to the manufacturer’s instructions. One colitis microgram total RNA was reverse transcribed using the GoScript reverse transcription system (Promega, Shanghai, China) following the supplier’s As expected, DSS damaged the colon’s mucosal barrier, leading to protocol. The reactions were incubated at 25˚C for 20 min, then at 42˚C for gut inflammation and weight loss. Mice in the DSS-treated group

Table I. Primers for real-time PCR

Gene Primer Sequences Product Size (bp) GenBank Accession No. TNF-a F: 59-GCATGGTGGTGGTTGTTTCTGACGAT-39 99 NM_010851.2 R: 59-GCTTCTGTTGGACACCTGGAGACA-39 IL-6 F: 59-GAGTCACAGAAGGAGTGGCTAAGGA-39 106 NM_031168.1 R: 59-CGCACTAGGTTTGCCGAGTAGATCT-39 IL-8 F: 59-TCTCGGTGTAGAGCAAGG-39 146 NM_011339.2 R: 59-TTCCCAAGTGCTGGTATT-39 zo-1 F: 59-TCATCCCAAATAAGAACAGAGC-39 198 XM_006540786.1 R: 59-GAAGAACAACCCTTTCATAAGC-39 zo-2 F: 59-GCTTTGGTGTGGACCAAGAT-39 269 XM_006526909.1 R: 59-TCCATTATGGGTTTGCATGA-39 claudin-1 F: 59-GCTGGGTTTCATCCTGGCTTCT-39 110 NM_016674.4 R: 59-CCTGAGCGGTCACGATGTTGTC-39 occludin F: 59-AAGCAAGTGAAGGGATCTGC-39 204 NM_001205255.1 R: 59-GGGGTTATGGTCCAAAGTCA-39 mucin-1 F: 59-TGGATTGTTTCTGCAGATTTT-39 147 NM_013605.2 R: 59-CCTGACCTGAACTTGATGCT-39 mucin-2 F: 59-CCCAGAAGGGACTGTGTATG-39 134 NM_023566.3 R: 59-TGCAGACACACTGCTCACA-39 GAPDH F: 59-CAACGGCACAGTCAAGGCTGAGA-39 112 NM_017008.3 R: 59-CTCAGCACCAGCATCACCCCAT-39 The sequences in Table I are available through GenBank (http://www.ncbi.nlm.nih.gov/nuccore/) under the accession numbers listed above. F, forward; R, reverse. The Journal of Immunology 1885 had significantly more weight loss than did those in the control than did those in the pBD2 group. Compared to the control group, group from days 4 to 7, whereas mice in the pBD2-treated group histological examination of the colon in the DSS group showed had less weight loss compared with mice in the DSS-treated extensive ulceration of the epithelial layer, edema, crypt damage of group. Mice in the pBD2 plus DSS group rapidly recovered the bowel wall, and infiltration of and mononuclear weight from days 4 to 7 (Fig. 1A). Similarly, mice in the pBD2 cells into the mucosa. Compared to mice in the DSS group, mice and pBD2 plus DSS groups had firmer stools and less blood in treated with pBD2 plus DSS had reduced histologic evidence of their stools than did those in the DSS group from days 1 to 7. DSS-induced colitis (Fig. 1D). DAI, consisting of weight loss, stool consistency, and OB, were Although DSS induced histological alterations only in the colon, measured daily (Fig. 1B). differences in immune cells and Igs could be detected between the Colon shortening was used as a marker of inflammation. Con- blood of control and DSS groups. To study the immune response in sistent with the DAI, by day 8, the colon length in the DSS group the blood, the concentrations of Igs in blood were analyzed. was shorter than that in the control group (4.10 6 0.22 versus Treatment with DSS decreased the concentrations of IgA and IgM, 5.61 6 0.13 cm, p , 0.05) and in the pBD2 plus DSS group but increased the concentration of IgG. There were improvements (4.10 6 0.22 versus 5.05 6 0.13 cm, p , 0.05) (Fig. 1C). Mice in in these parameters in mice from the pBD2 plus DSS group, the DSS group had significantly more macroscopic inflammation returning to levels closer to the control group (Fig. 1E, 1F). Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 1. pBD2 ameliorated clinical symptoms during the pathological process of DSS-induced colitis. (A) BW is shown as a percentage of daily weight change from days 0 to 7. (B) DAI is comprised of weight loss, stool consistency, and OB. (C) Quantitative measurement of the length of the colon. (D) Representative H&E-stained sections from (Da) control, (Db) DSS, (Dc) pBD2, and (Dd) DSS plus pBD2. Original magnification 3200. (E) Con- centrations of IgA, IgG, and IgM in venous blood. Results are given as means 6 SEM. Differences between groups were determined by ANOVA followed by Duncan’s test (n = 6). *p , 0.05 compared to control mice, #p , 0.05 compared to DSS-treated mice. 1886 b-DEFENSIN 2 ATTENUATES INJURY OF COLITIS

As depicted in Fig. 2A, there was a marked increase in MPO (an To determine the anti-inflammatory effect of pBD2 on DSS- indicator of colonic infiltration with polymorphonuclear leuko- induced colitis, the expression and secretion of the inflammatory cytes) and EPO (an indicator of colonic infiltration with eosino- markers TNF-a, IL-6, and IL-8 were evaluated in colon tissues. phils) activities in colon tissue from mice treated with DSS (162 6 Compared with the normal control group, there was a higher ex- 11 and 40.3 6 3.0 ng/g tissue for MPO and EPO, respectively) pression of TNF-a, IL-6, and IL-8 mRNA in the DSS-treated compared with control mice (34.9 6 10.0 and 30.9 6 1.7 ng/g group. However, the levels of TNF-a, IL-6, and IL-8 mRNA tissue, respectively). Administration of pBD2 to DSS-treated mice were significantly lower in both the pBD2- and pBD2 plus DSS– significantly decreased MPO and EPO activities (93.6 6 5.2 and treated groups compared with the DSS-treated group (p , 0.05) 22.6 6 1.2 ng/g tissue, respectively). (Fig. 3A). There was no significant difference between the pBD2- As shown by TUNEL staining, ,10 apoptotic cells per field and pBD2 plus DSS–treated groups. were found in the colon from all normal mice, or following Administration of DSS caused a significant elevation of colonic treatment with pBD2 alone. This number increased to 50 cells per levels of the proinflammatory cytokines TNF-a, IL-6, and IL-8 as field in the colon from the DSS-treated mice without pBD2 compared with those in the colons from the normal control group treatment. The administration of pBD2 significantly reduced the (Fig. 3B). Treatment of mice with pBD2 significantly reduced the number of apoptotic cells in DSS-treated mice (Fig. 2B). DSS-induced production of TNF-a, IL-6, and IL-8 compared with Infiltration of inflammatory cells (particularly CD177+ neu- that in the DSS alone group (Fig. 3B). Taken together, these data trophils and F4/80+ macrophages) into the colon is observed in indicated that treatment with pBD2 ameliorated the colonic in- acute colitis. In this study, the infiltration of CD177+ and F4/80+ flammation caused by DSS. cells into colonic tissue was detected by immunohistochemistry. Downloaded from There was minimal infiltration of (Fig. 2C) and pBD2 prevented the DSS-induced disruption of intestinal TJ macrophages (Fig. 2D) into the colons of control mice. Com- structure and function pared to control mice, DSS triggered an increased infiltration of To investigate the effect of pBD2 on intestinal structure and function, CD177+ neutrophils and F4/80+ macrophages into the colonic intestinal permeability was assessed using FITC-dextran perme- lesion area. Treatment with pBD2 decreased the infiltration of ability. Mice in the pBD2 plus DSS–treated group had a lower

neutrophils and macrophages compared with the group treated intestinal permeability to 4-kDa FITC-dextran when compared with http://www.jimmunol.org/ with DSS alone. that in the DSS alone–treated group (Fig. 4A). To evaluate the

FIGURE 2. pBD2 ameliorated the colonic

inflammation during the pathological process of by guest on September 25, 2021 DSS-induced colitis. Enzymatic activities of MPO (A) and EPO (B) were measured. Control and pBD2-treated groups were administered PBS or 5 mg/kg pBD2 rectally. DSS and DSS plus pBD2 groups received PBS or 5 mg/kg pBD2 rectally and 3.5% DSS orally. Rectal treatments were once daily from days 1 to 7. (C) TUNEL staining of colonic tissue. Original magnification, 3400. (Ca) Control, (Cb) DSS, (Cc) DSS plus pBD2, and (Cd) pBD2. (Ce) The difference in the number of apoptotic cells per field between DSS-treated mice and DSS plus pBD2–treated mice was statistically significant (p , 0.05). (D) Representative images of the infiltration of CD177+ cells. Original magnifi- cation, 3400. Formalin-fixed, paraffin-embed- ded 5-mm cross-sections were stained with a primary Ab to CD177. (Da)Control,(Db) DSS, (Dc) DSS plus pBD2, and (Dd) pBD2. (E) Rep- resentative images of the infiltration of F4/80+ cells. Original magnification 3400. Formalin- fixed, paraffin-embedded 5-mm cross-sections were stained with a primary Ab to F4/80. (Ea) Control, (Eb)DSS,(Ec) DSS plus pBD2, and (Ed) pBD2. Results are given as means 6 SEM. Differences between groups were determined by ANOVA followed by Duncan’s test (n =6). *p , 0.05 compared to control mice, #p , 0.05 compared to DSS-treated mice. The Journal of Immunology 1887

FIGURE 3. The effect of pBD2 on the transcriptional activation and secretion of proinflammatory genes. (A) Quantitative RT-PCR for TNF-a, IL-6, and IL-8 mRNA in colonic tissues. (B) ELISA for TNF-a, IL-6, and IL-8 in colonic tissues. Control and pBD2-treated groups received PBS or 5 mg/kg pBD2 rectally. DSS and DSS plus pBD2 groups received PBS or 5 mg/kg pBD2 rectally and 3.5% DSS orally. Rectal treatments were once daily from days 1 to 7. Results are given as means 6 SEM. Differences between groups were Downloaded from determined by ANOVA followed by Duncan’s test (n = 6). *p , 0.05 compared to control mice, #p , 0.05 compared to DSS-treated mice. http://www.jimmunol.org/

functional viability and integrity of mouse intestinal epithelium plus DSS–treated cells, the average TEER value was only mod- under ex vivo conditions, TEER measurements were performed for erately reduced by 2 h (Fig. 5A) and increased by 6 h, reaching 60 min. As shown in Fig. 4B, in the DSS alone–treated group, the .80% of the baseline value. At the end of the experiment (24 h) , TEER values declined significantly, indicating an increase in per- the average TEER value of the DSS-treated Caco-2 monolayer by guest on September 25, 2021 meability. In contrast, DSS-treated mice given pBD2 showed a remained significantly below the value of the control cells. In significant recovery in TEER. These data support a significant role contrast, the TEER value of Caco-2 cells treated with pBD2 plus for pBD2 activation in minimizing DSS-induced intestinal epithelial DSS was not significantly different from the baseline value or the hyperpermeability. value of the control cells. The expression of zo-1, zo-2, claudin-1, mucin-1,andmucin-2 Caco-2 cells form TJs when grown to a monolayer. To determine was monitored and compared between groups using real-time whether pBD2 could prevent the DSS-induced reduction in TJ PCR. As shown in Fig. 4C, all genes were downregulated in expression, real-time PCR analysis was performed for message mice treated with DSS alone, comparedwithnormalanimals. levels of TJ markers (Fig. 5B). DSS caused a significantly reduced Administration of pBD2 increased the expression of all genes up expression of zo-1, claudin-1, and occludin, compared with con- to 90% in DSS-treated mice. To further evaluate the protective trol (0.49 6 0.08 versus 1.00 6 0.03, 0.30 6 0.12 versus 1.00 6 effect of pBD2 on the disruption of TJs by treatment with DSS, 0.05, 0.61 6 0.03 versus 1.00 6 0.01, respectively; p , 0.05). The the TJ markers zo-1 and claudin-1 were detected by immuno- expression of TJ markers in the pBD2 plus DSS group were sig- fluorescence on the colon epithelium. In the control group, zo-1 nificantly higher than that in the DSS alone group (zo-1, 0.75 6 and claudin-1 proteins (red) were seen on the cellular membrane 0.06; claudin-1, 0.76 6 0.15; occluding, 0.93 6 0.07; p , 0.015) of the epithelial cells, mostly in the spinous and granular layers. (Fig. 5B). This supports the importance of pBD2 in maintaining In the pBD2 alone or with DSS-treated group, the expression of the integrity of the TJs. zo-1 and claudin-1 increased in both the cellular membrane and pBD2 inhibited the production of inflammatory mediators cytoplasm of spinous and granular layers in the mucosa. However, in the DSS-treated group, the expression of zo-1 and claudin-1 The effect of pBD2 on the proinflammatory mediators inducible decreased in individual cells of the spinous and granular layers NO synthase (iNOS) and cyclooxygenase-2 (COX-2) is presented (Fig. 4D, 4E). in Fig. 6A and 6B. Colonic iNOS and COX-2 levels in the DSS alone group were significantly higher (4.07 6 0.08 mM/100 mg pBD2 attenuated the DSS-induced paracellular permeability and 3.06 6 0.09 U/100 mg tissue, respectively) than the corre- in vitro sponding values in control animals (1.68 6 0.07 mM/100 mg and To further address the molecular mechanisms of the protective role 1.19 6 0.05 U/100 mg tissue, respectively). Treatment with pBD2 of pBD2 in DSS-induced changes to intestinal barrier function, we alone produced iNOS and COX-2 levels similar to the control used an established Caco-2 cell culture system and the TEER assay. group. The administration of pBD2 to DSS-treated mice signifi- When a monolayer of Caco-2 cells was incubated with 3.5% DSS, cantly decreased these inflammatory mediators (2.06 6 0.08 mM/ the TEER values rapidly decreased by 2 h and reached their lowest 100 mg and 1.31 6 0.04 U/100 mg for iNOS and COX-2, re- level (70% of the baseline value) after 4 h. However, in the pBD2 spectively). 1888 b-DEFENSIN 2 ATTENUATES INJURY OF COLITIS

FIGURE 4. Effect of pBD2 on intestinal TJ structure and function. (A) Quantification of serum FITC-dextran, a measure of intes- tinal barrier function. The levels of FITC- dextran were compared between the control, DSS, pBD2-treated, and pBD2 groups. (B) TEER of mouse colonic epithelium mea- sured ex vivo in Ussing chambers. Treatment with pBD2 attenuated the epithelial hyper- permeability induced by DSS. (C) Relative changes of gene expression of TJ proteins Downloaded from and mucins were determined by real-time PCR analysis. (Ca) zo-1, (Cb) zo-2, (Cc) claudin-1, (Cd) mucin-1, and (Ce) mucin-2. Immunofluorescence staining of zo-1 (D) and claudin-1 (E) for control (Da and Ea), DSS- (Db and Eb), DSS plus pBD2 (Dc and

Ec), and pBD2-treated (Dd and Ed) groups is http://www.jimmunol.org/ shown. Cells were counterstained with DAPI (blue). Original magnification, 3200. Immuno- fluorescent staining with anti–zo-1 (red) and anti–claudin-1 (red) Abs showed that both were markedly decreased in the DSS-treated group compared with the control and DSS plus pBD2–treated groups. Results are given as means 6 SEM. Differences between groups were determined by ANOVA followed by by guest on September 25, 2021 Duncan’s test (n =6).*p , 0.05 compared to control mice, #p , 0.05 compared to DSS- treated mice.

DSS caused a significant increase in the concentration of NO the phosphorylation of c-Fos (data not shown), a member of the in the supernatant of RAW264.7 cells. pBD2 significantly AP-1 family of proteins. This indicated that the inhibition was inhibited this DSS-induced increase in NO production (Fig. 6C). selective to NF-kB. Similarly, DSS significantly increased the production of PGE2 in RAW264.7 cells and 12.5 mg/ml pBD2 blocked this effect Discussion (Fig. 6D). Murine models of IBD have been used to investigate the regulatory mechanisms that reduce inflammation and restore intestinal ho- Effect of pBD2 on the in vitro activation of upstream signaling meostasis (33). We referenced the previous studies to determine k molecules for NF- B translocation the induction method of the colitis model (34–36) and a rough Immunohistochemistry results showed that LPS elevated the ex- dose range of peptide first (36–42). We found that providing pression of phosphorylated p65 NF-kB, IkB-a, and Akt compared drinking water containing DSS induces colitis was one of the with the control group (Fig. 7). Treatment with 12.5 mg/ml pBD2 well-established experimental models for studying IBD (43). The effectively inhibited the phosphorylation of these proteins. The present study focused on studying the effects of pBD2 on DSS- suppressive effect of pBD2 appears to involve NF-kB transloca- induced experimental colitis. We demonstrated that adminis- tion. Indeed, pBD2 suppressed the transcriptional regulation of tration of pBD2 effectively attenuated colonic inflammation in inflammatory genes caused by LPS by inhibiting the activation of mice with DSS-induced colitis. After 1 wk of treatment, BW p65, a major subunit of NF-kB. However, pBD2 did not suppress loss, DAI, colonic MPO activity, and macroscopic and histo- The Journal of Immunology 1889

FIGURE 5. Effect of pBD2 on DSS-induced paracellular permeability in vitro. (A) Effect of pBD2 (12.5 mg/ml) on TEER of a Caco-2 cell monolayer. Changes in TEER induced by DSS were prevented with pBD2. (B) Expression of zo-1, claudin-1, and occludin mRNA was as- sessed by real-time PCR. Treatment with DSS markedly decreased the expression of zo-1, claudin-1, and occludin mRNA compared with control. pBD2 prevented the reduction in TJ expression induced by DSS. Results are given as means 6 SEM. Differences between groups were determined by ANOVA followed by ,

Duncan’s test (n = 6). *p 0.05 compared to Downloaded from control cells, #p , 0.05 compared to DSS- treated cells. http://www.jimmunol.org/

logical changes were significantly improved in mice treated reduced the increased serum and colon levels of TNF-a,IL-6, rectally with 5 mg/kg/d pBD2. The present study also demon- and IL-8 caused by DSS. All of these findings suggest that pBD2 strated the anti-inflammatory and intestinal TJ regulatory ac- is protective in a mouse model of DSS-induced colitis. tivities of pBD2 against DSS-induced colitis. Additionally, we Colitis induced by DSS has characteristics similar to human have demonstrated that the molecular mechanisms involved in UC, such as signs of diarrhea, gross rectal bleeding, weight loss, the therapeutic effects of pBD2 on colitis involved an inhibition shortening of the colorectum, histological features of multiple by guest on September 25, 2021 of NF-kB signaling pathways. Finally, we showed that pBD2 erosions, and inflammatory mucosal changes occasionally in-

FIGURE 6. pBD2 inhibited the production of inflammatory mediators. Effects of pBD2 (12.5 mg/kg BW) on colonic iNOS (A) and COX-2 (B) in mice with DSS-induced colitis. Treatments were administered once daily for 7 consecutive days simultaneously with the induction of colitis. (C) Levels of NO determined by the Griess as- say in culture supernatants of RAW264.7 cells treated with LPS and/or pBD2 for 24 h. (D)

Levels of PGE2 determined by ELISA in culture supernatants of RAW264.7 cells treated with LPS and/or pBD2 for 24 h. Data are expressed as means 6 SEM of six independent assays. Dif- ferences between groups were determined by ANOVA followed by Duncan’s test. *p , 0.05 compared to control cells, #p , 0.05 compared to DSS-treated cells or LPS-treated cells. 1890 b-DEFENSIN 2 ATTENUATES INJURY OF COLITIS

FIGURE 7. Effect of pBD2 on the in vitro activation of upstream signaling molecules for NF-kB translocation. Phosphorylated and total protein levels of p65 NF-kB(A), IkB-a (B), Akt (C), and b-actin from RAW264.7 cell lysates were determined using Abs recognizing phospho-specific or total protein. Total protein levels of p65 NF-kB, IkB-a, Akt, and b-actin were not changed by treatment with LPS, pBD2, or pBD2 plus LPS, compared with control. However, 1 mg/ml LPS increased the expression of phosphorylated p65 NF-kB, IkB-a, and Akt. Treatment with pBD2 (12.5 mg/ml) inhibited the protein Downloaded from phosphorylation of p65 NF-kB, IkB-a, and Akt. cluding crypt abscess. The major causative factors in the initiation a physical and metabolic barrier against toxins and pathogens in of human colitis include enhanced vasopermeability and upregu- the lumen. The barrier regulates macromolecule trafficking be-

lation of inflammatory mediators and some cytokines. Prolonged tween the lumen and the internal milieu, and it protects the host by http://www.jimmunol.org/ infiltration is also involved in this animal model of preventing harmful solutes, , toxins, and luminal colitis (44, 45). Initiation and progression of intestinal inflam- Ags from entering the body (58). Compromising the barrier mation, including IBD, requires the presence of bacterial patho- function of the intestinal mucosa can result in an increased ex- gens (46, 47). To maintain normal homeostasis, the host has evolved posure of the host to luminal Ags and pathogens, leading to in- several antibacterial defense systems, including antimicrobial pep- flammation (59), and is a major contributing factor in multiple tides such as defensins, to defend against microorganisms (20). pathological conditions of the gastrointestinal tract. The epithelial Endogenous antimicrobial peptides play an important role in innate barrier function is largely determined by intercellular TJs. The TJs immunity. An important advance in the defensins field has been are responsible for restricting paracellular movement of com- evidence indicating that defensins may play a role in innate im- pounds across the intestinal mucosa (60, 61). An increased per- by guest on September 25, 2021 munity. Wehkamp et al. (48) reported an increased expression of meability in the TJ may provide a major site for both infection and colonic hBD2 mRNA in UC patients but not in CD patients. Altered the establishment of inflammation in the gut (62, 63). Bacterial defensin production was thus suggested to be an integral element in translocation is thought to occur via a paracellular pathway through the pathogenesis of IBD. the epithelial cells. Our data show that pBD2 might have a protec- Consistent with these findings, the present study provides tive effect on barrier integrity by maintaining the expression of TJs evidence for the involvement of defensins in the pathological and mucin proteins, thereby reducing the severity of gut inflam- process of colitis in DSS-exposed mice. b-Defensins comprise mation. The results of our study suggest that the bioavailability of a major subclass of the defensins (49) and play an important role pBD2 is an important contributing factor for determining epithelial in both innate and adaptive immune defense (50, 51). They are integrity. chiefly expressed in a variety of epithelial cells (e.g., airway Once the mucosal barrier is breached, the submucosa is exposed epithelia, urogenital tissues, nasolacrimal duct, and mammary to a vast pool of luminal Ags, including food and bacteria, thereby gland) and sometimes immune cells (e.g., dendritic cells and engaging the innate immune responses, including increased pro- macrophages) (50, 52, 53). Both humans and mice express duction of proinflammatory cytokines. We showed that the addition a relatively large number of b-defensins, of which six human of pBD2 ameliorated the increased expression in TNF-a and IL-6 (hBD1–6) and five mouse (mBD1–5) defensins have been iso- induced by DSS. The reduction in proinflammatory cytokines by lated and characterized. hBD1 and its murine ortholog mBD1 are pBD2 may be either due to a direct suppressive effect on their ex- expressed constitutively. Other b-defensins, including hBD2 and pression or effects on the maintenance of epithelial barrier func- its murine ortholog mBD3, are induced by bacteria and cyto- tion, leading to a reduction in foreign luminal antigenic load and kines. mBD3, similar to certain human b-defensins, possesses full activation of the . the ability to attract immune cells chemotactically (54). To date, A major function of defensins is thought to be protection of 12 pBDs have been identified, and the expression of pBD1 and the adjacent epithelial stem cells at the base of the crypt (64). pBD2 in the small intestine of the pig has been described re- Therefore, continual expression of defensins allows constant cently (55, 56). protection of the integrity of the epithelial barrier. However, as has From an immunological point of view, the epithelial lining of the been described in numerous studies, the expression of defensins intestine has been long considered as solely a physical barrier to can vary in the presence of disease (20). In the present study, the protect the underlying mucosa. Intestinal epithelial cells produce administration of pBD2 ameliorated signs and symptoms of in- cytokines and chemokines that are crucial for the recruitment and flammation, such as colon shortening, weight loss, and increased activation of immune cells. Additionally, they produce and secrete DAI. effector molecules, such as b-defensins, that can directly affect The infiltration of activated neutrophils is one of the most bacterial populations in the gut (57). The intestinal mucosa is prominent histological features observed in IBD (34, 65). Neu- The Journal of Immunology 1891 trophils produce superoxide anions and other reactive species elevated in the serum from mice with DSS-induced colitis. Im- leading to the formation of the very reactive hydroxyl radical that portantly, the administration of pBD2 significantly reduced the may contribute significantly to the tissue necrosis and mucosal serum levels of TNF-a and IL-6. These results indicated that dysfunction of IBD (66). In the present study, histological analysis pBD2 ameliorates DSS-induced colitis by suppressing proin- showed an increase in the colonic submucosal infiltration of flammatory mediators such as TNF-a and IL-6. NO is generated neutrophils and macrophages 7 d after mice were treated with enzymatically in a variety of cells by three isoforms of NO syn- DSS. Pretreatment with pBD2 in DSS-induced colitis significantly thase. Increased expression of iNOS leads to the synthesis of reduced the DAI, the wet weight of the colon (a reliable and micromolar quantities of NO that can damage cells through the sensitive indicator of the severity and extent of inflammatory re- formation of NO-reactive products and can be measured as a sponse) (67), and loss of BW. toxicity marker (82). The activation of COX-2 and iNOS pro- MPO is a peroxidase enzyme found in the azurophilic granules of duces excessive inflammatory mediators that may contribute to neutrophils and monocytes. The level of MPO activity is directly the development of intestinal damage. Additionally, iNOS acts proportional to the neutrophil concentration in the inflamed tis- in synergy with COX-2 to promote the inflammatory response sue and is thus an index of neutrophil infiltration and inflamma- (83). In the present study, NO and COX-2 were significantly tion (68). Therefore, the measurement of MPO activity has been increased in LPS-treated RAW264.7 cells. Treatment with pBD2 considered a quantitative and sensitive assay for assessing acute decreased NO production through downregulation of iNOS and intestinal inflammation. Our findings show that colonic MPO ac- also significantly diminished COX-2 expression. These effects tivity was markedly increased in DSS-treated mice, and that 7 d of may account for the protective role of pBD2 in the colitis model. treatment with pBD2 significantly reduced this effect. This sug- Similarly, earlier studies revealed that there was a reduced risk Downloaded from gests that pBD2 has an anti-inflammatory effect that parallels the of gastrointestinal ulcer complications using COX-2 inhibi- histological evidence of protection. tors (84). Apoptosis is one of the ulcerogenic processes in the gastric Proinflammatory cytokines such as IL-1b, IL-6, and TNF-a,as mucosa (69). Increasing evidence shows that dysregulation of this well as COX-2, iNOS, and adhesion molecules, are regulated process in the colonic mucosa is involved in the pathogenesis of through the NF-kB pathway (85). This suggests that blocking

IBD (70). As shown by TUNEL staining, DSS markedly increased these inflammatory mediators en masse by inhibiting NF-kB http://www.jimmunol.org/ the number of apoptotic cells in the colonic mucosa. Adminis- might be more effective in the treatment of colitis than sup- tration of pBD2 decreased the extent of apoptosis with no effect in pressing individual factors such as TNF-a and IL-6. The present the normal mucosa. Thus, one of the mechanisms for pBD2 to study revealed that the anti-inflammatory effect of pBD2 may be prevent DSS-induced colitis may be through an inhibition of ap- linked with the downregulation of p65 NF-kB in DSS-induced optosis in the colonic mucosa. colitis in mice. Western blot analysis indicated that the phos- The colonic epithelium is composed of trefoil peptides such as phorylation of p65 NF-kB was significantly increased by LPS in mucin-1 and mucin-2, in which mucin-2 is the predominant sec- RAW264.7 cells and was decreased after treatment with pBD2. retary mucin in the colon (71). The present study demonstrated that Similarly, the levels of TNF-a and IL-6 in the DSS-treated group mucin-1 and mucin-2 genes were markedly downregulated during were increased more than in the normal control group in response by guest on September 25, 2021 DSS-induced colitis. A dose of 5.0 mg/kg/d pBD2 significantly to p65 NF-kB. upregulated the expression of these genes at the mRNA level. A Because the upstream signaling events for NF-kB activation study with a mucin-2 knockout mouse model confirmed that have been well established, we examined specific targets of pBD2 mucin-2 deficiency caused abnormal morphology, ulceration of for the inhibition of NF-kB activity. As Fig. 7 depicts, pBD2 epithelial cells, and a mild increase of inflammatory cells in the suppressed the phosphorylation of IkB-a. The activation of up- colon (72). The knockout animals developed more serious colitis stream enzymes for IkB-a phosphorylation were also suppressed. symptoms by feeding 2.5% DSS for 2 d, suggesting that mucin-2 Thus, pBD2 reduced the phosphorylation of Akt. Because the is critical for colonic protection. The mucus layer in the colon of promoter regions of TNF-a and IL-6 contain consensus binding patients with UC was thinner than in healthy subjects (73), and motifs for NF-kB, our data strongly suggest that pBD2 can sup- a reduction of the mucus layer in UC patients was linked to press the production of inflammatory mediators such as NO and a decrease in mucin-2 expression (74). Taken together, pBD2 may PGE2 by suppressing NF-kB–mediated transcriptional activation be useful in treating colitis patients by boosting mucin gene ex- of inflammatory gene expression. pression and increasing mucus synthesis in the colonic mucosa, In summary, our data confirm that pBD2 prevents DSS-induced which is perturbed in UC patients. colitis in mice and that this protective effect may be due to UC is a nonspecific inflammatory disorder involving primarily maintaining the integrity of the intestinal mucosal barrier and re- the mucosa and submucosa of the colon. Although the precise gulating innate immunity. These effects are mediated via modula- etiopathogenesis of UC remains unknown, there is accumulating tion of TJ proteins in the colonic tissue, inhibiting the production evidence that an increase of proinflammatory mediators leads to an of inflammatory mediators (including iNOS, COX-2, TNF-a, and inflammation cascade and tissue damage (75). TNF-a, which is IL-6) and inhibiting NF-kB signal transduction pathways. Nu- a pleiotropic cytokine, plays a fundamental role in inflammatory merous efforts have focused on developing new anti-inflammatory conditions such as colitis by triggering the accumulation and ac- drugs targeted to NF-kB (86). The present results support previous tivation of leukocytes (76). Therefore, TNF-a is an important research suggesting that targeting NF-kB may be a new and ef- therapeutic target and several engineered Abs against TNF-a are fective treatment in IBD patients. beneficial in the treatment of IBD (77). IL-6 is produced by var- ious cell types and exerts pleiotropic effects on different organ Acknowledgments systems. Alterations in the production of IL-6 have been found in We thank Jinhui Li (Zhejiang University) for excellent technical assistance. inflammatory states such as rheumatoid arthritis (78), CD, UC (79), mesangial glomerulonephritis (80), and sepsis (81). IL-6 can stimulate neutrophil chemotaxis and lead to tissue destruction in Disclosures the colon. In the present study, the levels of TNF-a and IL-6 were The authors have no financial conflicts of interest. 1892 b-DEFENSIN 2 ATTENUATES INJURY OF COLITIS

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